Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation.

Lan T, Kisseleva T, Brenner DA - PLoS ONE (2015)

Bottom Line:
The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared.Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days).Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

Affiliation: Department of Medicine, University of California San Diego, La Jolla, California, United States of America; Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACTReactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX) play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as pro-fibrotic genes in hepatic stellate cells (HSCs) as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), or sonic hedgehog (Shh) in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days). Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

pone.0129743.g004: ROS production was attenuated by NOX1/4 inhibition in HSCs in response to Ang II stimulation.(A) HSCs from WT, NOX1KO and NOX4KO mice were loaded with H2DCFDA (10 μM) for 20 min. Cells were then washed and subsequently induced with Ang II (10-6 M). ROS production was assessed by fluorescent signals quantified continuously for 60 min using a fluorometer. (B) Representative images of ROS production in HSCs. Original magnification X10.

Mentions:
To identify the NOX members required for ROS generation in HSCs, we examined ROS generation in HSCs from WT, NOX1KO and NOX4KO mice. We quantitated the ROS generation in DCFDA-loaded HSCs after treatment with Ang II, a NOX agonist associated with liver fibrosis. Cells treated with buffer showed 10% increase in WT HSCs within 60 min, representing basal ROS production. In contrast, there was 4–5% increase in NOX1KO and NOX4KO HSCs (Fig 4A). Ang II induced 55%-60% increase in ROS production in WT HSCs, while there was a 40%-45% increase in NOX1KO and NOX4KO HSCs. In addition, Ang II induced higher DCFDA fluorescence in WT HSCs, but this fluorescence enhancement was suppressed in NOX1KO and NOX4KO mice (Fig 4B). These results suggest that both NOX1 and NOX4 mediate ROS production in HSCs in response to Ang II.

pone.0129743.g004: ROS production was attenuated by NOX1/4 inhibition in HSCs in response to Ang II stimulation.(A) HSCs from WT, NOX1KO and NOX4KO mice were loaded with H2DCFDA (10 μM) for 20 min. Cells were then washed and subsequently induced with Ang II (10-6 M). ROS production was assessed by fluorescent signals quantified continuously for 60 min using a fluorometer. (B) Representative images of ROS production in HSCs. Original magnification X10.

Mentions:
To identify the NOX members required for ROS generation in HSCs, we examined ROS generation in HSCs from WT, NOX1KO and NOX4KO mice. We quantitated the ROS generation in DCFDA-loaded HSCs after treatment with Ang II, a NOX agonist associated with liver fibrosis. Cells treated with buffer showed 10% increase in WT HSCs within 60 min, representing basal ROS production. In contrast, there was 4–5% increase in NOX1KO and NOX4KO HSCs (Fig 4A). Ang II induced 55%-60% increase in ROS production in WT HSCs, while there was a 40%-45% increase in NOX1KO and NOX4KO HSCs. In addition, Ang II induced higher DCFDA fluorescence in WT HSCs, but this fluorescence enhancement was suppressed in NOX1KO and NOX4KO mice (Fig 4B). These results suggest that both NOX1 and NOX4 mediate ROS production in HSCs in response to Ang II.

Bottom Line:
The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared.Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days).Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

Affiliation:
Department of Medicine, University of California San Diego, La Jolla, California, United States of America; Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACTReactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX) play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as pro-fibrotic genes in hepatic stellate cells (HSCs) as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), or sonic hedgehog (Shh) in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days). Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.